PROJECT SUMMARY/ABSTRACT The Reproductive Homeobox X-linked (Rhox) genes encode a novel family of transcription factors that are excellent candidates to regulate events during spermatogenesis and ovulation. The RHOX homeodomain protein family has several members (13 distinct genes, with some existing in multiple copies). However, only two are expressed in Sertoli cells, the master control cells that promote testis development and nurse the differentiation and survival of germ cells. Ablation of these two genes, Rhox5 and Rhox8, results in male subfertility characterized by lower epididymal sperm numbers, motility defects, and reduced fecundity. Progeny from Rhox5-null mice, crossed with RHOX8 in vivo RNAi knockdown mice possess a distinct phenotype that is more severe than either single mutant. While these two mouse lines are useful tools for discovering the role of RHOX5 and RHOX8 after puberty, they are incapable of shedding light on RHOX8?s potential role in gonad development as the inhibitory Rhox8 transgene does not turn on until after post-natal day 7 when androgen signaling normally turns on Rhox5. To address this issue, a new complete Rhox8 deletion model will be employed to assess RHOX8?s function in embryonic Sertoli cell specification and function. Characterization of these mice is of great interest because: 1) Rhox8 is the only Rhox gene expressed in Sertoli cells of the embryonic gonad. Thus, there will be no concerns of functional redundancy which has been an issue for Rhox studies in the past. All of the other members of the cluster are restricted to primordial germ cells, including Rhox5 which is only found in Sertoli cells postnatally. 2) Analysis of the Rhox8 postnatal in vivo knockdown mic, and transient knockdown of RHOX8 in embryonic testis cultures, show that RHOX8 regulates Sox9. 3) SOX9 is a master regulator downstream of SRY ? the factor that specifies male vs. female differentiation. Thus, RHOX8 may be in the sex determination pathway between SRY and SOX9. 4) After sex-determination, SOX9 governs multiple key events in the development of the tests including the formation of testis cords, male- specific vasculature, and differentiation of Sertoli and Leydig cell populations. If these post-puberty relationships are also conserved in the embryo, then RHOX8 may be a key cofactor or regulator of several events during gonad development. The experiments outlined in this proposal seek to systematically position Rhox8 in the milieu of male sexual development and discover the gene networks that are under its control. Because RHOX5 and RHOX8 may possess complementary functions in postnatal Sertoli cells, we will examine spermatogenesis in Rhox5/Rhox8 double knockout animals. Preliminary data suggests that these mice have a distinct and more severe phenotype than either single mutant. The elucidation of Rhox8?s function in Sertoli cells is important because it will provide an important ?building block? towards the long-term goal of learning the independent and collaborative functions of all the Rhox genes in the testes.